Composition and method for enhancing immune response

ABSTRACT

A composition and method for enhancing immune response in a living organism is disclosed. In particular, the present disclosure provides an adjuvant peptide for use in raising an immune response to an antigen. The adjuvant peptide is selected from a group of peptides with an HIV-related sequence. Additionally, the adjuvant peptide can comprise a fusion-protein that acts as a mucosal adjuvant. The adjuvant peptide can be transformed into one or more living cells, such that the mucosal adjuvant can be produced in living cells and then administered by systemic, mucosal or epidermal delivery.

CLAIM TO DOMESTIC PRIORITY

This Application is a U.S. National Stage Application filed under 35U.S.C. 371 claiming priority from the International Application No.PCT/US03/07073, filed Mar. 6, 2003, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/362,247, filed Mar. 6, 2002,and which applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to a composition and method forenhancing immune responses, and more specifically, to a composition andmethod using HIV-related peptides as an agent to increase immunogenicresponses and for delivering fusion proteins to animal cells.

BACKGROUND OF THE INVENTION

Most currently available vaccines consist of killed or live-attenuatedpathogens delivered by injection. Despite their success in preventingdisease, compelling conceptual, technical and economical reasons existto seek alternatives to traditional “Jennerian” vaccines.

Vaccines delivered parenterally require injections that must be given bymedically trained personnel. Additionally, injection risks possibletransmission of infection. Finally, parenteral delivery of vaccinesinvokes a systemic response, but not a mucosal response.

Subunit vaccines, especially those vaccines that target the mucosalimmune system, are viable, safe and effective alternatives. Muscosalvaccines do not require injection; thus, risk of transmission ofinfection is minimal. Finally, mucosal vaccines elicit immune responseboth systemically and mucosally.

Additionally, recent breakthroughs suggest that vaccines can be producedin edible tissues of transgenic plants that can then be orallyimmunogenic. The concept of using transgenic plants as vectors for theproduction and delivery of edible vaccines has been previouslydemonstrated.

However, to be effective, mucosal subunit vaccines often need to beco-administered with an “adjuvant.” An “adjuvant” is animmunostimulatory agent that would enhance the specific immune responsesagainst the vaccine candidate.

Therefore, a need exists for an immunostimulatory, mucosally-activecomposition that can be used as a systemic, mucosal, or epidermaladjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of a human immunodeficiency virus (HIV);

FIG. 2 depicts the structure of an adjuvant according to one embodiment;

FIG. 3 illustrates an ELISA determination of anti-CTB antibodiesfollowing immunization by gavage;

FIG. 4 shows end point titers of anti-CTB antibodies;

FIG. 5 illustrates reciprocal dilution of serum IgG₁.

FIG. 6 illustrates subclass titers of total serum IgG, IgG₁, IgG_(2a),IgG_(2b), IgG₃ and IgA.

FIG. 7 is a flowchart illustrating immune response of Th1 and Th2.

FIG. 8 depicts the synthesis of a plant-expression optimized DNAmolecule encoding for the P1 peptide;

FIG. 9 depicts maps of plasmids comprised of DNA sequences of CTB, P1,CTB-P1 fusion, and for the plant-expression of the CTB-P1 fusion;

FIG. 10 is a flowchart illustrating the construction of a CTB-P1 fusionprotein for plant-expression;

FIG. 11 depicts maps of plasmids for expression of CTB-P1 fusion proteinand CTB in tomato;

FIG. 12 shows the expression of CTB-P1 fusion protein in E. Coli cells;and

FIG. 13 illustrates an ELISA detection of anti-CTB and anti-P1 in E.Coli cells.

SUMMARY OF THE INVENTION

The present invention provides a composition and method for enhancingimmune response in living organisms, for example, in humans. In oneembodiment, and by way of example only, the composition includes, apeptide that when administered to a living organism, enhances theorganism's immune response. The composition may also include an antigen,for example, a cholera toxin. The composition may further include thepeptide and the antigen together as a fusion protein. The adjuvantpeptide may function as a systemic, mucosal or epidermal adjuvant.

In another exemplary embodiment, the adjuvant peptide may be encoded bya genetically-modified living cell. The genetically-modified living cellmay also encode an antigen. The peptide and antigen may also be encodedas a fusion protein.

Other independent features and advantages of the method for decreasingnicotine use in living organisms will become apparent from the followingdetailed description, taken in conjunction with the accompanyingdrawings which illustrate, by way of example, the principles of theinvention.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

This description discloses a composition and method for enhancing immuneresponse in living organisms by administering an oral, mucosal orepidermal adjuvant comprised of one or more HIV-related peptides.

FIG. 1 depicts the structure of an HIV retrovirus. HIV retrovirus 10 isan enveloped retrovirus. HIV retrovirus 10 is comprised of a viralmembrane 12, ampiphilic regions 14, charged helices 16, calcium (Ca²⁺)binding sites 18, gp41 subunits 20, and gp120 subunits 22. Adjuvantpeptide 24 facilitates HIV transcytosis across muscosal barriers towardthe serosal environment by binding to galactosyl ceramide (GalCer) onthe surface of mucosal epithelial cells.

Adjuvant peptide 24 comprises 36 amino acids (SEQ. ID. NO: 1)correspondes to a portion of the gp41 envelope. This peptide includes aconserved epitope (SEQ. ID. NO: 2), which is recognized by theneutralizing human IgG 2F5 and secretory IgAs that functionallyneutralize HIV transcytosis through epithelial cells. The conservedaromatic residues are important for GalCer binding.

FIG. 2 depicts the structure of an adjuvant 30 according to oneembodiment. Adjuvant 30 comprises peptides 32, linkers 34, and cargoproteins 36. However, alternate embodiments envision adjuvant 30comprising at least peptides 32, but not necessarily linkers 34 andcargo proteins 36. Peptides 32 may comprise adjuvant peptides as one ormore portions of P1 peptides, P5 peptides, or their functionalequivalents. In one embodiment, cargo protein 36 is an antigen, forexample cholera toxin. In an alternate embodiment, cargo protein 36 isany protein to be delivered to an animal cell.

According to one embodiment, an adjuvant peptide is a portion of the P1peptide, HIV envelope protein gp41, which includes the conservedepitope, lectin binding site (SEQ. ID. NO: 2). According to an alternateembodiment, the adjuvnat peptide is a portion of the P5 peptide, HIVenvelope protein gp41 which includes the P1 peptide and a calciumbinding site (residue number 622-684). P1 and P5 peptides also includetheir functional equivalents.

Functional equivalents of adjuvant peptides include peptides or portionsof larger proteins with overall sequence or structural similarity to P1or P5 peptides, and their derivatives, which allow the functionalitydisclosed here, including, but not limited to, one or more of thefollowing: enhancing the immune response, GalCer binding, binding to thesurface of cells containing GalCer, endocytosis to such cells ortranscytosis across a tight cell barrier.

Examples of functional equivalents include portions of variants of gp41in naturally occurring strains of HIV or in laboratory-derived strainsof HIV, including, but not limited to, site-directed mutated versions ofthe gp41 portion of the molecule. Specific, non-limiting, examples offunctional equivalents are HIV-1 isolate MN clone V5 (SEQ. ID. NO: 4),HIV-1 isolate 593 clone (SEQ. ID. NO: 5), HIV-1 isolate 98BRRS012 (SEQ.ID. NO: 6), and HIV-1 isolate 19242v3.20 (SEQ. ID. NO: 7).

Adjuvant 30 is capable of mucosal administration. Mucosal administrationincludes oral, nasal, vaginal, or rectal administration. Adjuvant 30 isalso capable of functioning as a systemic, mucosal, or epidermaladjuvant.

EXAMPLE 1

Example 1 demonstrates that adjvuant peptide enhances immune responsesagainst cholera toxin B subunit by mucosal co-administration of adjuvantpeptide and cholera toxin B subunit. Synthetic adjuvant peptide (SEQ.ID. NO: 3) with a C-terminal CONH₂, was synthesized by Eurogentec(Belgium) and by the Protein Chemistry Laboratory at Arizona StateUniversity. A cysteine residue was added to the beginning of SEQ. ID.NO: 1 to allow for dimerization (residue 649). Cholera Toxin B (CTB)subunit was chosen for co-administration because it is non-toxic and itis a strong mucosal adjuvant. Additionally, CTB binds to G_(M1)ganglioside whereby being able to target the fused antigen to mucosa.

Synthetic adjuvant peptide 30 micrograms (μg), adjuvant peptide plusCholera-Toxin B subunit (CTB) (30 and 70 μg, respectively), and CTB (70μg) were given orally to CD1 female mice (6-7 weeks old) by a gasticfeeding tube on day one, eight, and fifteen. The serum, fecal pelletsand vaginal secretions were collected prior to and on the second, thirdand fourth weeks after the first administration. Levels of anti-adjuvantpeptide and anti-CTB antibodies were determined by ELISA in each sample.

FIG. 3 illustrates an ELISA determination of anti-CTB antibodiesfollowing immunization by gavage of CTB (70 μg), CTB+P1 (70 μg and 30μg, respectively) or P1 (30 μg). Mice were gavaged on days indicated byarrows and samples of serum (A), fecal (B), and vaginal (C) secretionswere collected when indicated. Serum (A) detected systematic levels.Fecal (B) and vaginal (C) both detected mucosal levels.

Samples were serially diluted in phosphate buffered saline containing0.05% Tween-20 (PBST) containing 1% nonfat dry milk. Plates were coatedwith CTB overnight at 4° C., blocked with PBST containing 5% nonfat drymilk and then incubated with samples. Antibodies were detected byhorseradish peroxidase-conjugated secondary antiisotypic antiseraagainst the appropriate mouse antibodies (rabbit anti-mouse total IgGfrom CalBiochem, and the following anti mouse antisera: Anti-IgG₁,anti-IgG_(2a), anti-IgG_(2b), anti-IgG₃ from Santa Cruz Biotechnology;and anti-IgA from Sigma. FIGS. 3A-3C show maximal dilutions that allowedquantification.

FIG. 4 illustrates the end point of anti-CTB antibodies four weeks afterimmunization. Chemiluminescent ELISA was conducted as described in FIG.3. Titers in FIG. 4 are defined as reciprocals of the highest dilutiongiving a positive A₄₉₀ reading above 0.1. FIG. 5 illustrates, forexample, reciprocal dilution of serum IgG₁. FIG. 6 illustrates subclasstiters of total IgG, IgG₁, IgG_(2a), IgG_(2b), IgG₃ and IgA.

While in FIG. 4 antibody titers were below detection levels,co-administration of P1 and CTB to mice resulted in significantly highertiters of anti-CTB antibodies as compared to mice that were given CTBalone. Specifically, in FIG. 3, the level of fecal and vaginal anti-CTBIgA in the second and third week and serum anti-CTB in the second, thirdand fourth week appeared to be higher in mice fed P1 with CTB than inmice fed only CTB. Moreover, as illustrated in FIG. 6, co-administrationof P1 with CTB resulted in increasing all serum anti-CTB IgG subclass(IgG₁, IgG_(2a), IgG_(2b), IgG₃) titers by five to ten times in thefourth week, as compared to administration of CTB alone, as shown inFIG. 4.

Therefore, P1 peptide was shown to augment the production of mucosal IgAand serum IgG to co-administered CTB. Because CTB is a strong mucosalimmunogen by itself, the increase of both anti-CTB IgG₁, and IgG_(2a)levels suggest that the immune enhancement effect of P1 peptide isattributable to activating both Th1 and Th2 response. Th1 and Th2response is illustrated in FIG. 7. IgG_(2a) 40 effects T1 response 38through cell-mediated immunity, targeting intracellular pathogens 42.Antibodies 46, such as IgG₁ and IgA, effect Th2 response 44 targetingextracellular parasites, viruses and bacteria 48. Secondly, P1 peptidedid not induce antibody production against itself, even in the presenceof CTB. Therefore, P1 peptide can be used a mucosal adjuvant to enhanceimmune response in living organisms.

EXAMPLE 2

In example 2, plasmids were created for the co-expression of adjuvantpeptide and GFP in transgenic plants for oral delivery. FIG. 8 depictsthe synthesis of a plant-expression optimized DNA molecule encoding foran adjuvant peptide-GFP fusion protein. The sequence coding for adjuvantpeptide was inserted behind a DNA spacer encoding aGlycine-Proline-Glycine-Proline (GPGP) hinge. A BsrGI-SacI fragment ofthis plasmid was cloned in behind a 35 S Promoter. A PstI-EcoRI fragmentcontains the plant expression cassette. FIG. 8 represents a modeldelivery system for using fusion proteins to deliver cargo proteins toan animal cell.

FIG. 9 depicts maps of plasmids comprised of DNA sequences of CTB, P1,CTB-P1 fusion and for the plant-expression of the CTB-P1 fusion protein.A plant-expression optimized DNA molecule encoding for P1 peptide wassynthesized. The sequence was inserted behind a portion of the geneencoding the C-terminus of the CTB molecule behind a DNA spacer encodinga Glycine-Proline-Glycine-Proline (GPGP) hinge. An endoplasmic reticulum(ER) retention signal was engineered at the Carboxyl-Terminus. The PCRproduct was cloned into the cloning vector TOPO2.1 (Invitrogen) tocreate pTM058.

Still referring to FIG. 9, a HindIII-SacI fragment of this plasmid wasthen cloned into pTM042 to create a gene encoding a Carbosyl-terminusfusion of CTB and the P1 peptide in the plasmid pTM065. A BspHI-SacIfragment of this plasmid was cloned into pIBT210.1 (Haq, et al. 1995)behind a CaMV35S promoter and the 5′ UTR of Tobacco Etch Virus and infront of the 3′ UTR of the soy bean vspB gene to form pTM066. APstI-EcoRI fragment containing the plant expression cassette was clonedinto the T₁ plasmid derivative pGPTV-Kan (Becker, et al. 1992) to formpTM067 (not shown).

FIG. 10 is a flowchart illustrating the steps involved in creating aCTB-P1 fusion protein. In step 50, CTB (from HindIII site to the 3′end)-P1 fusion gene is designed and synthesized, a length of 234 basepairs (bp) (SEQ. ID. NO: 8). Next, in step 52, the CTB-P1 fusion gene iscloned into TOPO.

In step 54, the sequence is corrected by PCR-based site-directedmutagenesis to form pTM58 (pTM058). In step 56, the synthetic gene iscut out by HindIII and Sac I. Then in step 58, the synthetic gene iscloned into HindIII-SacI site of pTM42 (pTM042). This represents thecomplete CTB-P1 fusion gene.

The CTB-P1 fusion gene is then cut out by BspHI and SacI in step 60.Finally, in step 62, the cut out CTB-P1 fusion gene is cloned into theNcoI-SacI site of pTM38. Thus, step 62 clones the CTB-P1 fusion geneinto the plant expression cassette. The CTB-P1 fusion gene encodes theCTB-P1 fusion protein (SEQ. ID. NO: 9).

FIG. 11 illustrates an example for a construct for a potentiated ediblevaccine. In step 80, pTM086 containing the CTB-P1 fusion gene and theplant expression cassette was cloned into the T₁ plasmid derivativepGPTV-Kan (Becker, et al. 1992). The plasmid is then transformed intoAgrobacterium (LBA4404) in step 82. Finally, in step 84, theAgrobacterium is transformed into a tomato, for example MicroTom,cotyledon and hypocotyl explants.

In this example, the target organism for the adjuvant includes, but isnot limited to, Vibrio cholerae, enterotoxigenic Escherchia coli. Otherexamples would include virus-like particles, for example, Norwalk viruscapsid, and antigenic determinants of other pathogens, for example,bacterial, viral or parasitic.

EXAMPLE 3

The flowchart in FIG. 12 depicts purification protocol of CTB-P1 fusionprotein produced in E. coli. Resultant fractions from this protocol wereresolved by SDS PAGE on the right panel. The following were placed inthe first three lanes: Lane 1: molecular weight standards; Lane 2: amixture of denatured (lower monomeric band) and non-denaturedcommercially available CT-B (pentameric upper band); Lane 3: whole cellextract from an IPTG-induced E. coli.

Following sonication and centrifugations, in step 70, extracts areseparated into soluble (Lane 4) and insoluble (lane 5) fraction. Theinsoluble fraction, in step 72, is solubilized in 6.5 M urea andaffinity purification on nickel column in step 74. The eluate (Lane 6)is more than 90% pure and can be subjected to dialysis promoting therefolding and oligomerization of the monomeric CTB-P1 fusion protein. Byits mobility we conclude that the fusion protein can assemble intopentamers.

Finally, Eluate was dialyzed against PBS in step 76, and the purified,refolded CTB-P1 is shown in Lane 7. As noted in Lane 7, a CTB-P1pentameter was produced. Additionally, a CTB-P1 monomer with anintramolecular disulfide bond was also produced.

FIG. 13 demonstrates that the pentameric nature of the fusion proteinallows it to bind to G_(M1) gangliosides. The ELISA plate was coatedwith GM1-ganglioside. On the left half of the plate, anti-CTB was usedfor detection. On the left half of the plate, anti-P1 was used fordetection. CTB, CTB-P1 and P1 samples were applied to the plate asshown. The CTB is commercially available preparation of CTB and P1.CTB-P1 and P1 synthetic peptide are refolded samples purified asexplained in FIG. 12. Anti-CTB and anti-P1 are CTB- and P1-specificantibodies, respectively. These results demonstrate that the fusion isboth able to retain its pentameric structure as well as its P1 epitope.

Various embodiments of the invention are described above in the Drawingsand Description of Various Embodiments. While these descriptionsdirectly describe the above embodiments, it is understood that thoseskilled in the art may conceive modifications and/or variations to thespecific embodiments shown and described herein. Any such modificationsor variations that fall within the purview of this description areintended to be included therein as well. Unless specifically noted, itis the intention of the inventor that the words and phrases in thespecification and claims be given the ordinary and accustomed meaningsto those of ordinary skill in the applicable art(s). The foregoingdescription of a preferred embodiment and best mode of the inventionknown to the applicant at the time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in the light of the above teachings. Theembodiment was chosen and described in order to best explain theprinciples of the invention and its practical application and to enableothers skilled in the art to best utilize the invention in variousembodiments and with various modifications as are suited to theparticular use contemplated. Therefore, it is intended that theinvention not be limited to the particular embodiments disclosed forcarrying out this invention, but that the invention will include allembodiments falling within the scope of the appended claims.

1. A composition for enhancing immune response to an antigen in ananimal, comprising an isolated peptide consisting of an amino acidsequence, wherein the amino acid sequence is selected from the groupconsisting of SEQ. ID. NO: 1, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID.NO: 5, SEQ. ID. NO: 6, and SEQ. ID. NO: 7, and further comprising aheterologous an antigen.
 2. The composition of claim 1, wherein theantigen is cholera toxin.
 3. The composition of claim 1, wherein thepeptide and the heterologous antigen is a fusion protein.
 4. Thecomposition of claim 1, wherein the composition is capable of mucosaladministration.
 5. The composition of claim 1, wherein the compositionis formulated as a systemic adjuvant.
 6. The composition of claim 1,wherein the composition is formulated as a mucosal adjuvant.
 7. Thecomposition of claim 1, wherein the composition is formulated as anepidermal adjuvant.
 8. A method of enhancing immune response to anantigen in an animal comprising: providing a composition of claim 1 tothe animal.
 9. The method of claim 8, wherein the antigen is a choleratoxin.
 10. The method of claim 9, wherein the peptide and theheterologous antigen is a fusion protein.
 11. The method of claim 8,wherein the step of administering the peptide is carried out mucosally.12. The composition of claim 3, wherein the fusion protein comprises theprotein of SEQ ID NO:9.
 13. The method of claim 10, wherein the fusionprotein comprises the protein of SEQ ID NO:9.